Antibodies in therapeutic use are being developed so they have an increased proportion of ‘human’ or ‘humanized’ features. Antibodies are composed of heterodimers of an immunoglobulin light chain and heavy chain. The two chains in combination dictate the antigen recognition properties of the antibody directed by the ‘variable’ regions of each chain. The antibody preserves the combinatorial recognition features to the antigen, yet may be delivered therapeutically as an altered, engineered, processed, or fragment polypeptide molecule, such as an antibody tetramer, Fab or (Fab)2, or as an immunoconjugate or fusion protein with other polypeptide and chemical entities providing additional properties.
Fully human antibodies are important therapeutic proteins. In these examples, human antibodies have been formed from a number of screening methods. These features have been created by generating new human antibodies as described in the examination of phage display library screening, and the use of immunization of transgenic mouse strains with embedded human antibody genes in place of the mouse antibody genes. These methods focus on the new derivation of human antibodies with similar antigen-binding properties to the non-human antibody. In cases where comparative analysis is possible between non-human antibodies directed against an antigen with those generated by either of the above methods, it is apparent that antigen binding affinity is ordinarily reduced. In addition these methods are limited by the utilization of incomplete human repertoire and antibody maturation capacity. Therefore, these methods suffer from requirements for continued refinements by recombinant engineering methods once a functional, but unsatisfactory human antibody is identified. Furthermore, these methods make no accommodation for the acquisition of immunogenic features of the new antibodies.
Methods are also in use to transform non-human monoclonal antibodies into therapeutic proteins without derivation of a new antibody. Humanized antibodies have been described that have improved properties indicating a reduced immunoreactivity in patients, and thereby making those reagents more useful for therapy, especially in prolonged exposure to the patient. Changes to the composition of the antibody that have been utilized are ‘CDR grafting’, a procedure where mouse or rat monoclonal antibodies are converted to another form where human framework substitutions are combined with the rodent CDR regions by molecular engineering approaches. A refinement of this strategy involves the recruitment of human variable gene sequences. U.S. Pat. No. 6,180,370 (the entire teaching of which is incorporated herein by reference) describes humanization strategies based on DNA sequence alignment between a parent antibody and similar human variable chains as an approach to replicate the antigen binding properties of a parent non-human antibody. This method does not specify the computational techniques necessary to evaluate the atomic coordinates of the antibody subfeatures, nor does it comment on the means to influence immunogenicity of component parts of the antigen-recognition features.
Since the source of the immunogenicity of non-human antibodies in human therapeutics is recognition by the human immune system of foreign protein sequences in the antibody polypeptides, an approach to reduce immunogenicity would be to reduce the amount of non-human protein sequence in a modified antibody while retaining those protein sequences that are essential for the antigen binding specificity of the parent antibody. The first of these alterations to antibodies to be employed were termed chimeric antibodies. The modifications consisted of replacement of the constant region domains of the parent antibody chains with human constant region domains, thus reducing the amount of non-human sequence by approximately half. However, these antibodies were shown to still have significant immune liabilities in the clinic (Hwang and Foote, 2005, Methods Vol. 36, p 3-10 and references therein, the entire teachings of which are incorporated herein by reference).
Other methods were subsequently developed to remove even more of the non-human sequence from the resultant modified antibody. These antibodies were termed humanized antibodies. In the method of complementarity determining region (CDR) grafting (U.S. Pat. No. 5,225,539 and Jones et al., 1986; Nature 321:522-525, the entire teachings of which are incorporated herein by reference), only those protein sequences of the parent antibody predicted to be essential for antigen binding are retained. The identity of these CDR sequences is first predicted from biochemical and x-ray crystal structure analyses of many antibodies, mostly derived from a mouse source (Al-Lazikani et al., 1997; Journal of Molecular Biology 273:927-948, the entire teaching of which is incorporated herein by reference). There are three CDR sequences in each chain of the antibody heavy and light chains. These six CDR sequences are grafted into equivalent sequence environments in a human antibody framework. This modified humanized antibody therefore only contains parental sequence in approximately 75 amino acid residues, which is a greater reduction of non-human sequence than the chimeric antibodies. Limitations of this method were revealed because the human framework chosen in humanization of a particular antibody may not be compatible with proper folding of the parent CDR sequences. An additional problem associated with these methods is that there are significant differences between mouse and human CDR, particularly in the heavy chain CDR3 (Zemlin et al., 2003; Journal of Molecular Biology 334:733-749, the entire teaching of which is incorporated herein by reference).
The method of Queen et al. (U.S. Pat. No. 6,180,370) further refines the process of CDR grafting. In this method, the human framework antibody is chosen by sequence homology to the parent antibody. In this way, the chances of proper folding are increased in the modified construct since the residues contacting the parent CDR sequences (Vernier residues) are more likely to be the same. Other contacting residues can be identified for modification in the framework region by identification of non-conserved residues in the sequence alignment of the parent antibody and the framework antibody as well as homology modeling of the parent and framework antibodies.
An even further reduction in the amount of parent antibody sequence can be achieved by using a method called specificity determining residue (SDR) grafting (Tamura et al., 2000; J. Immunol. 164:1432-1441, the entire teaching of which is incorporated herein by reference). In this method, CDR residues of a CDR-grafted, humanized antibody are systematically mutated and then analyzed for both ligand affinity and reactivity to sensitized sera samples. Once identified, the SDR residues important for binding are maintained, while those that are immunogenic are mutated. This method suffers from the need to experimentally identify the SDR residues.
An alternate method of reducing immunogenicity in the humanized antibody is modification of residues in the non-human antibody sequence that are predicted to be immunogenic but not critical for antigen binding based on solvent-exposed sites or folds (U.S. Pat. No. 5,869,619, the entire teaching of which is incorporated herein by reference). Others have mapped the solvent-accessible surfaces of antibodies by computer-instructed modeling (Zhang et al., 2005; Molecular Immunology 42:1445-1451, the entire teaching of which is incorporated herein by reference). These approaches have been utilized in combination with immunogenicity computer methods to delineate residues for recombinant engineering purposes.
In an alternative approach, a method (U.S. Pat. No. 6,881,557, the entire teaching of which is incorporated herein by reference) is used where the CDRs of the parent antibody are compared in amino acid sequence to candidate human CDRs to identify antibodies with most closely matched CDR loops. The residues in the human CDR region are then substituted with residues from the parent CDRs based on the linear alignments. This method is limited by the ability to match CDR epitopes without consideration of the framework residues, and does not incorporate three dimensional configurations of the CDRs.
The CDR grafting methods described above all rely on amino acid sequence information to determine which non-human residues should be grafted into the specific human acceptor antibody. Success of this technique resulting in a modified antibody with the desired antigen binding properties requires that the parental sequences are able to fold in three dimensions such that the spatial relationship of the key antigen binding residues is conserved between the parent and the modified structures. This being the case, utilizing information from the structure of the parent and potential human template antibodies should enable better decisions about which sequences from the parent antibody should be modified in the final antibody.
For instance, in the method described in U.S. Patent application 20040133357 (the entire teaching of which is incorporated herein by reference), the protein structure information of the parent antibody is solely used to guide assignment of an alignment of amino acid sequences of human antibodies. From these alignments, the amino acid variation at each position in the sequence is tabulated. Through modeling and energy minimization, the list of variants is filtered to include those variants that constitute a combinatorial library of antibody sequences. The method relies on screening for desired properties since the combinatorial libraries formed are large. Also, no structural criteria are elaborated and there is no computational refinement of this sequences based on additional protein structure considerations.
Another method utilizes homology models of the parent and human antibodies ( Luo et al., 2003: Journal of Immunological Methods 275:31-40, the entire teaching of which is incorporated herein by reference). In this method, a model was created of the murine parent as well as a human framework based on a consensus sequence used as a framework. The CDR structures in the human model were replaced by the corresponding murine model CDR structures. Through energy minimization calculations, residues important for CDR folding that were not optimized in the human framework were indicated. These residues were then altered in the model and then in the sequence to murine equivalents to improve the antigen binding properties of the modified antibody.
The structure can be even more informative if it is used to determine the exact human antibody frameworks with the best chance for successful grafting of parent CDR sequences. Yazaki et al. (Yazaki et al., 2004, Protein Engineering, Design & Selection, vol. 17, pp. 481-489, the entire teaching of which is incorporated herein by reference) outlined a structure-guided method for humanization of antibodies. Human or humanized antibody heavy chain or light chain structures that superposed well with the parental framework and CDR structures, were used as scaffolds for CDR grafting. A second filter of sequence identity was used to refine the selection, and energy minimization calculations identified residues for changes to alleviate unfavorable contacts. This method has the critical limitation of only aligning single protein chains to other single protein chains without consideration of quaternary structure. Importantly, the antigen binding site is made up from correct spatial arrangement of six CDR sequences from both the heavy and light chains. However, the structural information was not used to guide potential further reductions in the non-human sequence content of the grafted CDR.
The need for protein structure in humanization of antibodies is now evident for several reasons. Analysis of the amino acid sequences does not predict the folded protein state adequately, because small changes in the structure of the antigen binding site may result in large losses in affinity. Also, residues that are predicted to be solvent exposed may well be buried or critical for folding. It is evident that the most homologous human framework antibody by sequence alignment may not be the best scaffold for humanization. Although CDR loops superpose well in various structures when considered individually, the superposition is poor when evaluating the entire antibody chain (Bajorath et al. 1995; Journal of Biological Chemistry 270:22081-22084, the entire teaching of which is incorporated herein by reference). Further, large movements of the CDR positions arise from small changes in the junctional regions of the antibody. It is difficult to anticipate appropriate junctional residues and side chains based on sequence homology/alignment and evolutionary relatedness alone. Therefore, methods that incorporate the analysis of the three dimensional positioning of atoms in antibody variable regions will be the best representations and provide the most effective template for changes in antibody engineering. A method is needed to find the best three dimensional scaffold on which to build the antigen binding site, regardless of sequence variation.
Whereas protein structural information is a key advancement in the humanization process, improvements to the current methods are clearly necessary. A method is required that extends the current analysis in CDR grafting such that the two domains of the modified antibody are correctly oriented with respect to one another. In this way, the best human scaffold in terms of structure can be chosen for the CDR grafts, which should result in better conservation of the folded state of the CDR sequences after grafting, and improved affinity and specificity of the humanized antibody. A unifying method is needed for the consideration of the quaternary structure in the modification method in order to conserve the similarity to parent antibodies while improving the desired properties in the modified antibody. The overall folding of the modified antibody can be assumed to occur as independent elements for the framework and six CDR regions. Since the CDR fold locally into distinct classes, small changes between structurally similar loops are unlikely to disrupt the local folding of the loop. Thus, the structure can also be used to guide incorporation of a minimal amount of parent sequence into a human CDR sequence or framework region while maintaining the correct local folding of the loop as well as the affinity and selectivity for the antigen. The modified loops and framework may properly orient themselves with respect to one another, forming a binding site whose structure is conserved from the parent antibody, once fully assembled into a final structure containing elements from as many as seven human and one non-human structure. Additionally, structural information can be utilized to guide selection of residues for conservation of the binding properties of the modified antibody as well as guiding the selection of residues that can be mutated to form a library of potential humanized constructs that may have improved properties over the initial construct.